Background: Cutaneous lipids, 50% of which are ceramides (Cer), have structural and signaling roles in skin. The current study is based on our previous finding that biofilm infected wounds may appear visually closed but remain functionally open because of lack of barrier function of the repaired skin. Such defectively closed wounds display high trans-epidermal water loss (TEWL). The objective of this study was to test whether cutaneous ceramide depletion following wound biofilm infection compromises skin barrier function. Methods: Full thickness burn wounds (2”x2”) were created on the dorsum of pigs and followed up to 56 days post-wounding with and without infection with mixed bacterial species consisting of Pseudomonas aeruginosa and Acinetobacter baumannii, or Pseudomonas ceramidase mutant strains. Analyses of wound closure (digital planimetry), skin barrier function (TEWL), ceramide levels [lipidomics, using electrospray ionization-mass spectrometry (ESI/MS), immunohistochemistry (IHC)], PPARδ, ABCA12, loricrin expression (IHC and quantitative RT-PCR), and Nile Red fluorescent staining for skin lipid distribution were performed. Results: Bacterial ceramidases were over expressed (~500 fold, n=4, p<0.05) in biofilm-infected pig wound tissues. Immunohistochemical studies demonstrated that biofilm-infection caused focal erosion of ceramides in the wound-edge of porcine skin. Lipidomic analyses showed that long chain ceramides were depleted in biofilm-infected porcine skin wounds. Depletion of cutaneous ceramides downregulated the transcription factor PPARδ resulting in compromised transport of lipid molecules to stratum corneum by ABCA12, a lipid transporter. Decreased PPARδ expression further lowered the expression of the keratinocyte differentiation marker, loricrin. Conclusion: Excessive microbial ceramidases in biofilm infected wounds deplete host skin ceramides. Such disruption of ceramide homeostasis in the skin causes compromised barrier function of the largest organ in the body.